Most cells in the body possess a single primary cilium. These cilia are key transducers of chemical stimuli. Indirect evidence suggests that transduction of chemical stimuli by the primary cilium often depends on ion-conducting channels expressed in the ciliary membrane. However, the tiny size of the cilium has been a critical barrier to understanding its chemosensory functions. Existing studies of the ciliary channels have been indirect, using exogenous expression systems, artificial bilayers, and non-ciliary cellular compartments. Studies in the native ciliary membrane are virtually non-existent. To address this limitation, the applicants have developed a novel method that allows sensitive, repeatable, and stable electrical recordings from single primary cilia of cultured kidney cells. Recording begins the instant the cilium is plucked from the cell. The central goal of this project is to demonstrate that this is a highly efficient means of identifying mechanisms of electrochemical transduction in primary cilia. There are two specific aims. The first aim is to demonstrate that electrochemical transduction of external stimuli relevant to renal physiology can be effectively studied by recording from the cilium. The second aim is to demonstrate that ciliary recording is an efficient way to learn the effects of second messengers and voltage changes on ciliary transduction channels. This aim is proposed because preliminary studies by the applicants have identified ligand- and voltage-gated channels in the primary cilia. Because primary cilia are present on most mammalian cells, the significance of this work will be broad. In the kidney, mutations in ciliary proteins, including at least one channel protein, are hypothesized to cause cystic diseases that affect over 6,000,000 individuals worldwide. Validation of the novel method as proposed will reveal the functional physiology of the renal primary cilium in a way that has not previously been possible. Ultimately this will facilitate a new field of research: direct studies of the chemotransduction properties of the many primary cilia in the body. Such studies may identify targets for pharmacological intervention in treating diseases caused by defects in primary cilia. PUBLIC HEALTH RELEVANCE: Defects in the primary cilium underlie a wide range of human diseases, including cystic kidney diseases, cancer, and obesity. The work proposed here will advance understanding of the mechanisms by which primary cilia contribute to health and disease, particularly in the kidney. This will advance the NIH mission to pursue fundamental knowledge that will reduce the burdens of human illness.